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. 2015 Sep 15;195(6):2552-9.
doi: 10.4049/jimmunol.1501097. Epub 2015 Aug 3.

Th Cell Diversity in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

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Free PMC article

Th Cell Diversity in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

Kevin S Carbajal et al. J Immunol. .
Free PMC article

Abstract

Multiple sclerosis (MS) is believed to be initiated by myelin-reactive CD4(+) Th cells. IL-12-polarized Th1 cells, IL-23-polarized Th17 cells, and Th17 cells that acquire Th1 characteristics were each implicated in autoimmune pathogenesis. It is debated whether Th cells that can drive the development of demyelinating lesions are phenotypically diverse or arise from a single lineage. In the current study, we assessed the requirement of IL-12 or IL-23 stimulation, as well as Th plasticity, for the differentiation of T cells capable of inducing CNS axon damage. We found that stable murine Th1 and Th17 cells independently transfer experimental autoimmune encephalomyelitis (widely used as an animal model of MS) in the absence of IL-23 and IL-12, respectively. Plastic Th17 cells are particularly potent mediators of demyelination and axonopathy. In parallel studies, we identified MS patients who consistently mount either IFN-γ- or IL-17-skewed responses to myelin basic protein over the course of a year. Brain magnetic resonance imaging revealed that patients with mixed IFN-γ and IL-17 responses have relatively high T1 lesion burden, a measure of permanent axon damage. Our data challenge the dogma that IL-23 and Th17 plasticity are universally required for the development of experimental autoimmune encephalomyelitis. This study definitively demonstrates that autoimmune demyelinating disease can be driven by distinct Th-polarizing factors and effector subsets, underscoring the importance of a customized approach to the pharmaceutical management of MS.

Figures

FIGURE 1
FIGURE 1
IL-12 and IL-23 polarized CD4+ T cells are both capable of mediating ON. (A-C) C57BL/6 mice were immunized with MOG35-55 emulsified in CFA. Ten to fourteen days later, draining lymph node cells were harvested and cultured for 96 hours with MOG35-55 and either recombinant IL-12 or IL-23 to generate Th1 and Th17 cells, respectively. (A) Intracellular staining and flow cytometric analysis of cultured cells at 96 hours, gating on the CD3+CD4+ population. (B, C) Following culture, CD4+ T cells were transferred into naïve syngenic recipients. Mice in each group were euthanized at day 9 post-transfer, the day after clinical EAE onset. Mononuclear cells isolated from optic nerves (ON) and spinal cords (SC) were assessed for CD4+ T cell cytokine production (B) and the percent of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes (C) by flow cytometry. Data are representative of three independent experiments with at least 3 mice per group.
FIGURE 2
FIGURE 2
Axonopathy is an early feature of both Th1- and Th17-mediated ON. (A–D) Th1 and Th17 adoptive transfer recipients were injected i.o. with fluorochrome-conjugated cholera toxin-B (red) at onset of clinical EAE. Optic nerves were harvested 24 hours later. Longitudinal sections were stained for the pan-leukocyte marker, CD45 (green) to demarcate areas of inflammation. (C) Confocal microscopy revealed a swelling at the tip of a transected axon (asterisk). (D) The frequency of axonal swellings was compared with the intensity of CD45 staining and there was no significant difference. (E–G) Cross- sections of optic nerves harvested from mice with Th17- (E) and Th1- (F) mediated ON were stained for unphosphorylated neurofilament-H (SMI-32, green; DAPI, blue; bars=50 μm). Magnified views of the insets are shown in adjacent panels (bars=25 μm). (G) SMI-32 staining of a healthy nerve (bar= 50 μm). (H–J) Representative cross-sections of optic nerves obtained from mice with Th17- (H) or Th1-(I) mediated ON, or from naïve mice (J), were stained with toludine blue. Broken lines delineate representative areas with normal-appearing axons, asteriscs indicate swollen axons and arrowheads point to empty myelin sheaths (bars= 25 μm).
FIGURE 3
FIGURE 3
Axon loss and faulty nerve conduction occur in both Th1- and Th17-mediated ON. (A–D) Optic nerves harvested immediately before the peak of EAE were stained with antibodies against SMI32 (green) and CD45 (red). Representative cross-sections of nerves from mice with Th17- (A) and Th1- (B) mediated ON, as well as from a naïve mouse (B), are shown (bars = 100 μm and 25 μm, inset) and quantified (D). (E) Brn3a+ RGCs were counted in retinas harvested from naïve mice, and from ON mice at serial time points post-transfer (t-test). (F) Representative traces of CAPs of optic nerves acutely isolated from naïve and ON mice. CAP velocities (G, I) and amplitudes (H, J) were averaged over seven to twelve nerves per group. Data presented as mean ±S.E.M. (**p<0.01, ***p<0.001, ****p<0.0001; Mann-Whitney) Scale bars: A, B 50μm, insets 25μm; C 25μm.
FIGURE 4
FIGURE 4
Stable Th17 cells induce ON. (A) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-12KO mice, after 96 hours of culture with MOG35-55 and recombinant IL-23. (B) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-12KO donors after infiltrating the spinal cords and optic nerves of IL-12KO hosts. (C) Clinical courses of IL-12KO mice injected with myelin-specific CD4+ Th17 cells derived from IL-12KO donors versus WT mice injected with WT effector Th17 cells. The data shown is pooled from 3 experiments with n=10 WT and n=17 IL12KO host mice per group. (D) The percentage of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes infiltrating the optic nerve and spinal cord of IL-12KO hosts shortly after the onset of clinical EAE. (E) Contiguous sections of an optic nerve obtained from an IL-12KO host at clinical EAE onset and stained with CD45 (red, top left panel) or SMI-32 (green). (F) A representative section stained with toludine blue (bars= 50μm in D, 25μm in E). Areas with normal appearing axons are outlined, asteriscs indicate swollen axons and arrowheads point to empty myelin sheaths. (G–I) CAPs were measured in acutely isolated optic nerves from the IL-12KO recipients of stable Th17 cells at clinical EAE onset. (G) Representative wave-forms of optic nerve CAPs from a naïve IL-12KO mouse and an IL-12KO adoptive transfer recipient with acute ON. The data were averaged over seven to nine nerves per group. Data are presented as mean ±S.E.M. (*p<0.05, **p<0.01; Mann-Whitney)
FIGURE 5
FIGURE 5
Bona Fide Th1 cells are capable of inducing ON. (A) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-23KO mice, after 96 hours of culture with MOG35-55 and recombinant IL-12. (B) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-23KO donors after infiltrating the spinal cords and optic nerves of IL-23KO hosts. (C) Clinical courses of IL-23KO mice injected with myelin-specific CD4+ Th1 cells derived from IL-23KO donors versus WT mice injected with WT effector Th17 cells. The data was pooled from 3 experiments with n=9 WT and n=22 IL-23KO hosts (*p<0.05, **p<0.01; Holm-Sidak multiple t-tests). (D) The percentage of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes infiltrating the optic nerve and spinal cord. (E) Contiguous sections of a representative optic nerve obtained from an IL-23KO host at clinical EAE onset and stained with CD45 (red, left panel) or SMI-32 (green, right panels). (F) A representative section stained with toluidine blue. (bars= 50μm in E, 25μm in insets and in F). Examples of areas with normal appearing axons are outlined, asterisks mark examples of swollen axons and arrowheads point to myelin sheaths left behind by degenerated axons. (G–I) CAPs were measured in optic nerves from the IL-23KO recipients of Th1 cells at clinical EAE onset. The data were averaged over seven to nine nerves per group. (G) Representative wave-forms. CAP velocities (H) and amplitudes (I) were measured in eight to twelve nerves per group. Data are presented as mean ± S.E.M. (*p<0.05, **p<0.01; Mann-Whitney)
FIGURE 6
FIGURE 6
MS patients have diverse myelin-specific cytokine profiles. (A–C) PBMC were collected from relapsing MS patients with moderate disability on a monthly basis over the course of 1 year. (A) ELISPOT assays were performed to assess the frequency of MBP-specific IFNγ and IL-17 producers. Representative examples are shown. Patients were classified into three groups: IFNγ predominant (n=11), IL-17 predominant (n=7) and IFNγ/IL-17 mixed (n=18), based on criteria described in the Materials and Methods section. (B, C) Each subject underwent cerebral MRI scanning. T1- and T2-weighted lesion volume and brain parenchymal volume (BPV) were measured using a semi-automated approach [17]. The figures show average T1 and T2 lesion volumes normalized to total BPV. (*p=0.02; non-parametric, one-way ANOVA with Dunn’s multiple comparisons test)

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